Biological effects of cerium oxide nanoparticles. Implications at the Bio-Nano interface

Résumé

The nanotechnology and nanoscience fields have evolved sharply during last years. Many different nanomaterials have been synthesized and used in numerous products and consumer goods and many more will come shortly into the market. This revolutionary technology brings plenty of advantages in many sectors and, at the same time, it generates several uncertainties that need to be tackled regarding the unintended risks and potential harmful effects to human health and the environment. The pathways of entry into the environment, the biological effect to the whole food chain and the potential fate of nanomaterials in specific environmental compartments are just some of the open questions that need to be answered before nanotechnology continues growing. Recently, cerium oxide nanoparticles (CNPs) have received considerable attention due to their wide range of applications, their unique surface chemistry and their antioxidant/oxidant duality. However, a deep characterization of their bioactivity and interrelationship with other molecules has not been fully undertaken yet. The overall aim of this Thesis was to address the biological effects of cerium oxide nanoparticles (CNPs), emphasizing the role played by the Bio-Nano interface. Chapter 1 constitutes a general introduction, which includes the description of some basic concepts such as nanotechnology, nanomaterials and their implications. The particularities of CNPs derived of their physicochemical properties are described: It also contains a section dedicated to examine their interactions with different molecules and environmentally relevant organisms. Within Chapter 2, the state of the art of the Bio-Nano interface field is presented from an environmental point of view. Nanomaterials, once in the environment, are prone to suffer alterations (chemical, physical and biological transformations) which affect the Bio-Nano interface, so a full description of those transformations is included. Moreover, the importance and impact of the formation of an environmental identity or eco-corona onto the surface of nanomaterials is discussed and highlighted. The Research Needs section discusses the problems and knowledge gaps that need to be resolved in the near future regarding the environmental Bio-Nano interface. The biological effects and toxicological mechanisms of different types of CNPs (varying morphologies, sizes, coatings and methods of synthesis) towards two environmentally relevant microorganisms (the microalgae Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii) are presented and discussed in Chapters 3 and 4. Chapter 3 evaluates the importance of several particle properties such as nominal size, effective diameter, % surface Ce3+, nanoparticle ζ-potential and shape as explanatory variables for the observed bioactivity. The results show that only one driver, the percent (ratio) of surface Ce3+, explains the toxic response. The toxicological mechanisms of CNPs are the formation of abiotic Reactive Oxygen Species (ROS) and the attachment of particles to the cell envelopes. A totally different mechanism is observed when the CNPs are coated with an organic molecule (Chapter 4). Polyvinylpyrrolidone(PVP)-CNPs increased the formation of intracellular ROS, which impaired some physiological parameters without direct damage to cell envelopes. Different degrees of toxicity are found depending on the PVP chain length: The higher the molecular weight, the lower the toxicity observed. Little is known about the internalization process of CNPs in algae. There is evidence of CNP-internalization by some organisms, but the internalization mechanism and route of uptake are not fully understood. In Chapter 4, it is described that all CNPs are able to internalize in the microalgal cells and that the process follows a time dependent relationship. Besides, using various endocytosis inhibitors, it is determined that the clathrin-dependent endocytosis is the main pathway for nanoparticle entry. Due to the coexistence of oxidant and antioxidant properties in CNPs, the results presented in Chapters 3 and 4 shed light regarding the contradictory biological effects of CNPs that have been reported in the scientific literature. The reported discrepancies on the effects of CNPs could be attributed to the fact that the surface content of Ce3+/4+ has not been measured in all cases and also that, the biological effect of coated-CNPs has been disregarded. Chapters 5 and 6 are dedicated to study the interaction between CNPs and two different molecules. Firstly, the antioxidant properties of CNPs are expanded by proving that CNPs are able to scavenge in vitro hypochlorite anions (a strong oxidant compound) (Chapter 5). Surface interaction and the reduction of Ce4+ to Ce3+ revealed as the scavenging mechanisms. Secondly, the interactions of several CNPs with a 1,8-naphthalimide derivative are also studied (Chapter 6). CNPs could modulate the photophysical properties of the molecule as their spectroscopic properties varied depending on the complex formed. The bioluminescent model bacterium (Anabaena sp. PCC7120 CPB4337) allowed assessing the effect of CNPs and the complex they form with the organic molecule (nahthalimide derivative). The complex between the organic molecule and CNP with the higher content of surface Ce3+ acted additively towards the used model organism. Conversely, when the complex was formed by CNPs with higher content of surface Ce4+, an antagonistic effect was observed, highlighting the importance of CNPs surface chemistry. Altogether, this Thesis shows that CNPs are a versatile kind of nanomaterial whose biological effects could be extremely different depending on the physicochemical characteristics derived from their synthesis. The adsorption of molecules on their surface could modulate their Bio-Nano interface resulting in reduced toxicity. The surface content of Ce3+/Ce4+ and the type of coating must be taken into account by safer-by-design strategies, in the Environmental Health and Safety assessment of CNPs, but also in specific applications where a precise content should be presented to display their intended function. As CNPs have shown solid harmful effects towards several environmentally relevant microorganisms, this Thesis aimed at improving our understanding of the toxicity of this type of nanomaterial and by these means to contribute to the sustainable and reasonable development of nanotechnology.